The phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates concentrative uptake of sugars across the inner membrane in bacteria by a unique mechanism, using phosphorylation of the sugar substrate in concert with transport to drive uptake against the concentration gradient. The PTS comprises several soluble phosphotransferases and an integral membrane protein, EIIC. The EIIC component is responsible for substrate specificity, translocation across the membrane, and is necessary for the phosphorylation reaction to proceed. We recently solved the X-ray structure of an EIIC homolog, ChbC, in an inward-occluded conformation, which has allowed us to propose hypotheses for the conformational changes that bring the substrate across the bilayer and for how the phosphorylation reaction occurs. Using the unique resources and expertise provided by the member labs and core facilities of the MPSDC, we intend to use a variety of structural, spectroscopic, and computational techniques to test these hypotheses and build a comprehensive model of the structural dynamics of the EIIC transport cycle. Specifically, we will combine X-ray crystallography, DEER EPR, smFRET, and molecular dynamics simulations to determine the structures of the outward-open and inward-open conformations of the transporter, and to identify how EIIC forms a ternary complex with its sugar substrate and the soluble PTS component EIIB in order to catalyze the phosphorylation reaction.